1. Field of the Invention
The present invention relates to a fusion protein of human epidermal growth factor(xe2x80x9chEGFxe2x80x9d) and human angiogenin, and a process for preparing the fusion protein, more specifically, to a fusion protein of hEGF that tracks down the cancer cells expressing hEGF receptors at high level following internalization and angiogenin that exhibits cytotoxicity by degrading ribonucleic acids upon internalization, a process for preparing the fusion protein which employs E. coli transformed with an expression vector encoding a gene for the fusion protein, and its therapeutic application as an anticanacer agent.
2. Description of the Prior Art
Environmental pollution and increase in population of old-aged people cause increase of the rate of cancer by 5% every year. Cancer ranks first as the major cause of death among other diseases and accidents.
Chemotherapy has been widely used as an effective way to prevent and cure cancer. However, it has been known that chemotherapy provokes various side-effects, e.g., attacking the normal cells as well as the cancer cells. Due to low specificity for the cancer cells and toxicity for the normal cells, numerous attempts have been made to develop anticancer agents with high specificity and low toxicity. The examples of such attempts include search for the drug with novel mode of action, development of drug delivery system and drug targeting, and use of a secondary agent to aid activity of a primary anticancer agent.
Recently, a new method of drug targeting has been studied that is selectively active to the cancer cells by using a chemical agent bound to a carrier which has strong affinity to the cancer cells. As a promising resolution, it has been reported that antibodies against to antigens specific to the cancer cells, such as alpha-fetoprotein, are bound to the chemical agents, ricin, Diphtheria toxin, Pseudomonas toxin, or radioisotopes(see: K. Shikoro et al., Br. Med. Bull., 40:233-239(1984); Vijay et al., Nature, 339:394-397(1989); U.S. Pat. No. 4,545,985; Peter et al., Cancer Res., 54:1008(1994)). However, the antibody-bound chemical agents have some shortcomings, such as difficulty in penetrating the cells, antigenicity of antibody itself, binding to the normal cells, low binding efficiency due to relatively high molecular weight of antibody(see: Pastan et al., Cell, 47:641-648(1986); Hurwitz et al., Cancer Res., 35:1175-1181(1975); Delabye et al., J. Clin. Invest., 77:301-311(1986); Buchegger et al., J. Exp. Med., 158:413-427(1986); Buchegger et al., Cancer, 58:655-661(1986)).
Another attempt has been made to develop an anticancer agent using polyamino acids as a carrier since polyamino acids do not exhibit toxicity to the normal cells. After the agent forms a complex by binding to the polyamino acids, it is detached from the complex by the enzymes present in the cancer cells. However, this strategy failed to solve the aforementioned problems(see: Kato et al., J. Med. Chem., 27:1602-1607(1984); EP 112,720; U.S. Pat. No. 4,485,093).
As one of attempts for anticancer drug targeting, formation of a protein complex between a toxic protein and a peptide or, a protein capable of binding to the intracellular receptors has been extensively studied, though it has not been fruitful. Such peptides or proteins are TGF(tumor growth factor), MSH(melanocyte stimulating hormone), somatostatin, glucagon, insulin, transferrin, LDL(low density lipoprotein), calcitonin, alpha-2-macroglobulin, bradykinin, and EGF(see: U.S. Pat. No. 4,545,985; Shimizu et al., FEBS Letters, 118:274-278(1980); Cawley et al., Cell, 22:563-570(1980); Simpson et al., Cell, 29:469-473(1982); W085/00369; W083/04030; U.S. Pat. No 4,528,186; EP 46,039; EP 128,733; W085/01284; EP 131,868; U.S. Pat. No. 3,917,824).
Also, genetically engineered fusion proteins of peptides and cytotoxins have been generated by binding Diphtheria toxin to TRH(thyrotropin releasing hormone), TRF(transferrin), MSH, and LDL. Usage of these fusion proteins, however, has been restricted due to difficulties in maintaining the receptor binding activity of carrier proteins(see: Bacha et al., J. Biol. Chem., 258:1565-1570(1983); Okeefe et al., J. Biol. Chem., 260:932-937(1985); Murphy et al., Proc. Natl. Acad. Sci., USA, 83:8258-8262 (1986); Japanese Patent Publication No.(Sho)60-163824).
Toxicity of a protein complex of EGF and a cytotoxic chemical has not been lowered due to toxicity of the cytotoxic chemical itself and immunotoxicity resulted from antibody formation against the chemical(see: WO88/00837). EP 467,536 describes that genetically engineered TGF-fused to modified Pseudomonas exotoxin A can be used to target bladder cancer. However, it failed to overcome antigenicity of the toxin derived from the microorganism. Japanese Patent Publication No.(Sho) 63-41418 discloses a protein conjugate of EGF and Pseudomonas toxin using a chemical material as a linker. Again, toxicity issue raised by Pseudomonas toxin has remained to be solved. EP 11111 suggests an anticancer agent which is prepared by chemical conjugation of a low molecular weight growth factor with a chemical which binds non-specifically to carboxyl terminal or xcex2-amino terminal of the protein. The prior art is, however, proven to be less satisfactory in the senses of contamination during chemical reaction, denaturation of the protein, and heterogeneicity of the conjugate itself.
As mentioned above, research on the cytotoxic agents or microorganism-derived toxins fused to monoclonal antibodies recognizing molecules on the surface of the cancer cells(see: U.S. Pat. No. 4,664, 911; U.S. Pat. No. 4,545,985) or studies on the fusion proteins generated by fusing a growth factor originated from neoplastic tissue and a microorganism-derived toxin(see: Jill et al., J. Biol. Chem., 266:21118(1991); Daniel et al., Cell, 22:563-570(1980); Caudhary et al., Proc. Natl. Acad. Sci., USA, 84:4538-4542(1987)) are still ongoing, however, they have not been fruitful since these studies still need to overcome the problems caused by toxicity of the proteins, chemical contamination, immunotoxicity resulted from antibody formation, non-specific binding of antibodies, difficulty in delivering a high molecular weight fusion protein to the target cells(see: Chung et al., Mol. Cells, 6:125-132(1996)).
The present inventors have made an effort to solve the aforementioned problems of the immunotoxicity resulted from antibody formation and the low targeting efficiency due to a high molecular weight of at least 50 kD, and finally developed a genetically engineered low molecular weight fusion protein consisting of hEGF and angiogenin both of which normally exist in human body and then exhibit no toxicity following overdose administration. Further, the inventors manufactured a large quantity of the fusion protein by cloning and expressing a fusion gene encoding hEGF and angiogenin in bacteria, whose efficacy is substantially improved in comparison with the conventional anticancer agents in the senses that: 1) it selectively inhibits the growth of the cancer cells expressing hEGF receptor; 2) it does not have a detrimental effect on the growth of the normal cells; 3) it does not exhibit toxicity of the conventional chemical anticancer agents; and, 4) it does not cause any serious problem by forming antibody against the fusion protein.
A primary object of the present invention is, therefore, to provide a fusion protein of hEGF and angiogenin that selectively inhibits growth of the cancer cells expressing hEGF receptors.
The other object is to provide a process for preparing the fusion protein by employing recombinant microorganism transformed with an expression vector containing the gene encoding the fusion protein.
Another object is to provide an anticancer agent comprising the fusion protein as an active ingredient.
The above and the other objects and features of the present invention will become apparent from the following description given in conjunction with the accompanying drawings, in which:
FIG. 1 is a plasmid map of an expression vector pTE105.
FIG. 2 is a schematic diagram depicting construction strategy of a recombinant vector pRSang.
FIG. 3 is a schematic diagram depicting construction strategy of a recombinant vector pTEang.
FIG. 4 is a schematic diagram depicting construction strategy of a recombinant vector pTE4081 that expresses a fusion protein of angiogenin-glycine-hEGF.
FIG. 5 is a schematic diagram depicting construction strategy of a recombinant vector pTE4082 that expresses a fusion protein of angiogenin-(glycine)4 serine-hEGF.
FIG. 6 is a schematic diagram depicting construction strategy of a recombinant vector pTE4083 that expresses a fusion protein of hEGF-angiogenin.
FIG. 7 is a schematic diagram depicting construction strategy of a recombinant vector pTE4084 that expresses a fusion protein of hEGF-glycine-angiogenin.
FIG. 8 is a schematic diagram depicting construction strategy of a recombinant vector pTE4089 that expresses a fusion protein of hEGF-(glycine)4 serine-angiogenin.
FIG. 9 is a schematic diagram depicting construction strategy of a recombinant vector pTE40810 that expresses a fusion protein of hEGF-[(glycine)4 serine]2-angiogenin.
FIG. 10 is a schematic diagram depicting construction strategy of a recombinant vector pTE40815 that expresses a fusion protein of angiogenin-(glycine)4 serine-hEGF-(glycine)4 serine-angiogenin.
FIG. 11 is a schematic diagram depicting construction strategy of a recombinant pTE40816 that expresses a fusion protein of hEGF-(glycine)4 serine-angiogenin-(glycine)4 serine-hEGF.
FIG. 12(A) is a photograph of bacterial culture of E.coli JM 101 transformants analyzed by SDS-PAGE technique.
FIG. 12(B) is a photograph of Western blot analysis of FIG. 12(A).
FIG. 13(A) is a photograph of SDS-PAGE and Western blot analysis of bacterial culture of E.coli JM 101 transformed by a recombinant vector pTE4089.
FIG. 13(B) is a photograph of SDS-PAGE and Western blot analysis of bacterial culture of E.coli JM 101 transformed by a recombinant vector pTE40810.
FIG. 14(A) is a photograph showing the size of the tumors in control mice.
FIG. 14(B) is a photograph showing the size of the tumors in mice injected with hEGF-(glycine)4 serine-angiogenin.